Ribozymes are RNA molecules having enzymatic activity. They have been voluntarily implicated in gene expression and control, such as RNA splicing, RNA processing, RNA genome replication and ribosomal peptide-bond formation. In particular, the group I intron ribozyme from Tetrahymena thermophila can join together two exons flanking the intron via the natural cis-splicing, and also can perform trans-splicing reactions to join two exons contained on separate RNAs. The trans-splicing proceeds via a cleavage-ligation reaction through a two-step transesterification mechanism. The ribozyme recognizes and binds a separate substrate RNA (5′ exon) by forming base pairs between the target RNA and its internal guide sequence (IGS). Once bound, the ribozyme cleaves the targeted RNA, releases the downstream cleavage product and splices an exon sequence at its 3′ end onto the 5′ exon cleavage product of the substrate RNA instead of a cleaved 5′ exon RNA region of its self-splicing process. This splicing occurs in E. coli and mammalian cells, as well as in vitro. The trans-splicing reaction can be applied for targeting and cleaving a specific target RNA and replacing it with a desired RNA sequence. Thus, trans-splicing ribozymes have the potential as novel therapeutic genes, which are capable of repairing defective RNA transcripts associated with genetic diseases with normal RNAs, or of targeting a specific RNA to express a desired RNA only within cells expressing the target RNA.
For example, a group I intron-based trans-splicing ribozyme has been shown to convert sickle βs-globin transcripts into mRNAs encoding the anti-sickling protein γ-globin in erythrocyte precursors (Lan, N., et al., Science 280: 1593-1596. 1998). Also, a trans-splicing ribozyme was employed to amend a mutant myotonic dystrophy protein kinase transcript and a transcript of a mutant canine skeletal muscle chloride channel, causing myotonia congenita (Phylactou, L. A., et al., Nat. Genet. 18: 378-381, 1998; Rogers, C. S., et al., J. Clin. Invest. 110: 1783-1798, 2002). A trans-splicing ribozyme has been reported to induce the death of cancer cells by repairing mutant p53 transcripts with wild-type p53 transcripts (Shin, K. S., et al., Mol. Ther. 10: 365-372, 2004), and to block the replication of hepatitis C virus (HCV) by recognizing a specific region of HCV RNA genome and replacing it with RNA expressing anti-viral activity (Ryu, K. J., et al., Mol. Ther. 7: 386-395, 2003). Recently, many studies involve the potential of trans-splicing ribozymes as therapeutic genes for genetic diseases. For example, the expression of a hTERT-targeting group I intron-based trans-splicing ribozyme has been shown to induce the selective cytotoxicity in tumor cells expressing human telomerase reverse transcriptase (hTERT), and to have effective anticancer activity in an animal model implanted with human carcinoma cells (Kwon, B. S., et al., Mol. Ther. 12: 824-834, 2005). However, there are no reports describing the application of ribozymes as biosensor molecules and imaging agents for screening specific ligands and for use in the diagnosis of diseases. The present invention is the first to establish the above use of ribozymes.
Precise diagnosis of diseases is essential for successful gene therapy. Early diagnosis in particular is very critical for the successful treatment of diseases. Imaging technology and chemical and biological markers have been used for the early diagnosis of diseases, especially cancer. However, diagnostic imaging is disadvantageous in that it is unable to be used in the early stages. Chemical and biological markers exhibit low diagnostic accuracy, and thus, standard methods for early diagnosis using these markers have not been established. In particular, for gene-associated diseases, such as cancer, since there are no methods capable of monitoring the expression of associated genes in vivo, diseases can be detected when the body's response to diseases occurs only after an inordinate amount of time has passed since the associated genes began expression. Thus, diseases such as cancer are rarely curable in their early stages. Also, since the progression of diseases or responses to treatment after surgery or therapy is impossible to be monitored in the early stages in which gene expression occurs and can be monitored only when visual responses occur in the body, it is very difficult to perform a treatment which is optimal for individual patients. Various imaging methods, such as X-ray, CT, MRI, SPECT, PET and sonography, have been used for diagnosing cancer or genetic diseases. An image of a target site is assessed to determine whether it is cancerous in practice through pathological evaluation of a tissue sample isolated using invasive biopsy. However, when a tissue biopsy cannot be taken, interpretation of the image is difficult. Also, for treatment, tumor excision should be performed when a tumor increases to a size in which it can be excised thorough a surgery, thereby delaying diagnosis and not enabling proper treatment at the early stages.
Molecular imaging is a new approach that visualizes, through imaging, various molecular events occurring in cells, that is, gene expression, biochemical processes and biological changes. Molecular imaging enables doctors to determine the onset of cancer or other diseases in images of target tissues, and thus allows the non-invasive monitoring of suitable early treatment and non-surgical treatment without tissue biopsy. A composition for molecular imaging and a molecular imaging method according to the present invention have advantages overcoming disadvantages of existing molecular imaging technology, and harbor location signal accuracy and high temporal resolution, which will be needed in gene therapy and imaging technology in the future. Also, the composition and method of the present invention have beneficial clinical applications because a material used in molecular imaging does not have harmful side-effects, which it has not been possible yet to eradicate from molecular imaging employing quantum dot nanoparticles, which have been studied as a future technology.